Therapeutic use of p75ntr neurotrophin binding protein

ABSTRACT

The present invention relates to a new therapeutic use of a p75NTR neurotrophin binding protein and related molecules in the treatment of osteoarthritis.

FIELD OF THE INVENTION

The present invention relates to a new therapeutic use of a p75NTRneurotrophin binding protein and related molecules in the treatment ofosteoarthritis.

BACKGROUND TO THE INVENTION

Osteoarthritis is a group of conditions in which the joints aredegraded, leading to pain, tenderness, stiffness and locking of joints.It is the most common form of arthritis, affecting millions of peoplearound the world.

The condition is generally accepted to be the result of mechanicaldamage to cartilage in joints with inadequate self repair. In additionto leading to inflammation, pain and swelling, the loss of cartilage canlead to the formation of bone outgrowths (osteophytes) which exacerbatesymptoms and can lead to narrowing and distortion of the joint. Theprecise mechanisms of cartilage damage and loss are not preciselyunderstood and may be the result of a combination of factors.

Treatment of osteoarthritis is limited to management of the condition,with no curative treatment options available. There are also no reportedtreatments which halt progression of this degenerative disease.Management options include physiotherapy, administration of pain killersand/or administration of anti-inflammatory drugs and, in some cases,joint replacement via surgery.

The neurotrophins, neurotrophic growth factor (NGF), brain-derivedneurotrophic factor (BDNF), neurotrophin 3 (NT-3), and neurotrophin 4/5(NT-4/5) act via four receptors: the low affinity p75 neurotrophinreceptor (p75NTR), and the high affinity tyrosine kinase receptors;TrkA, TrkB, and TrkC. The low affinity receptor p75NTR binds and isactivated by all four neurotrophins and has been reported to functionindependently from the other receptors. However, the Trk receptors aremore selectively activated i.e. NGF is the selective ligand for TrkA,BDNF the ligand for TrkB and NT-3, 4/5 the ligands for TrkC. In additionit has been reported, when p75NTR and Trk proteins are co-expressed,they form complexes, which alter the signalling of both receptors (Huangand Reichardt, 2003). Indeed, it has been suggested that p75NTRfacilitates the selectivity of each of the neurotrophins for theirrespective Trk receptor.

The p75NTR is a member of the tumor necrosis factor receptor superfamily(TNFR-SF) and was the first member of this superfamily to becharacterized fully. The superfamily (encoded by some 30 genes inhumans) is defined by ligand-binding domains consisting of one or more(typically four) repeats of a 40 amino acid cysteine-rich domain (CRD)that was first identified in p75NTR (Johnson et al., 1986; Radeke etal., 1987). In contrast, no sequence motif is shared by theintracellular domains of all TNFR-SF family members. Consequently,signalling mechanisms of TNFR-SF proteins vary significantly.

An unusual feature of p75NTR structure is the existence of adisulfide-linked p75NTR dimer, formed via cysteinyl residues within thetransmembrane domains. This disulfide linkage is required for effectiveneurotrophin-dependent signalling by p75NTR and plays an important rolein the formation of an intracellular and extracellular domain (Vilar etal., 2009b). Neurotrophins exist physiologically as noncovalentlyassociated dimers (Bothwell and Shooter, 1977) with a distributionhalf-life of approximately 5 min (Tria et al., 1994).Neurotrophin-dependent p75NTR activation involves association of aneurotrophin dimer with CRDs 2-4 of the two extracellular domains of ap75NTR dimer (He and Garcia, 2004). Recent studies support a model inwhich neurotrophin binding causes the two extracellular domains ofp75NTR dimers to move closer together, forcing the intracellular domainsto splay apart in a snail-tong-like motion centered on the disulfidebond and permitting association of the intracellular domains with thesignalling adapter proteins, NRIF and TRAF6 (Vilar et al., 2009a,2009b). Intra-transmembrane domain disulfide bonds, such as are presentin p75NTR, have not been described previously in other TNFR-SF familymembers, or in any other membrane protein.

p75NTR undergoes sequential proteolytic cleavage by alpha-secretase andgamma-secretase activities and matrix metalloproteinases (MMPs),releasing its intracellular domain (ICD) into the cytoplasm, in a manneranalogous to the cleavage-dependent signalling pathway of Notch andbeta-amyloid precursor protein (Jung et al., 2003; Kanning et al.,2003). Cytoplasmic release of the p75NTR ICD by this pathway promotessignalling by associated NRIF (Kenchappa et al., 2006). The role of theextracellular domain of p75NTR, following the proteolytic cleavage byalpha-secretase and gamma-secretase activities and MMPs is not fullyunderstood.

It has been documented that NGF and other neurotrophins (BDNF, NT-3 andNT-4/5) play a significant role in pathology for example pain due toosteoarthritis, pancreatitis, rheumatoid arthritis, psoriasis, pruritisand multiple sclerosis (Watanabe et al., 2010; Raychaudhuri et al.,2011; Barthel et al., 2009; Truzzi et al., 2011; McDonald et al., 2011;Yamaoka et al., 2007). It was been demonstrated that selectiveantibodies to any of the neurotrophins; either NGF or BDNF, NT-3 andNT-4/5 significantly reduce pain. Furthermore, antibodies directed tothe neurotrophin receptors p75NTR Trk A, Trk B or Trk C have also beendemonstrated to be efficacious in models of pain (Orita S et al., 2010;Svensson P et al., 2010; Iwakura et al., 2010; Cirilio et al., 2010;Pezet et al., 2010; Hayashi et al., 2011; Chu et al., 2011; Ueda et al.,2010; Ghilardi et al., 2010; Fukui et al., 2010). Fukui et al., (2010)in a model of pain (mechanical allodynia following sciatic nerve crush)demonstrated significant efficacy on pain related endpoints followingtreatment with an anti-p75NTR antibody. It was concluded from this studythat the treatment with a p75NTR inhibitory antibody reduced CGRP andp75NTR expression resulting in a significant reduction in pain.

BRIEF DESCRIPTION OF THE INVENTION

The current invention demonstrates the extracellular domain of p75NTR isuseful in the treatment of osteoarthritis. The extracellular domain ofp75NTR has been shown to halt and even reverse the progression of thedisease.

Accordingly, there is provided in a first aspect a p75NTR neurotrophinbinding protein (p75NTR(NBP)) for use in the treatment ofosteoarthritis.

In preferred embodiments, the treatment of osteoarthritis includesrelief from the symptoms of osteoarthritis. Preferably relief from thesymptoms of osteoarthritis include, but are not limited to reduction inpain, inflammation, swelling, tenderness, joint stiffness or increase injoint mobility or any combination of these.

In a particularly preferred embodiment, treatment of osteoarthritisincludes slowing or arresting of disease progression and/or reduction incartilage loss. Preferably treatment of osteoarthritis includes reversalof disease progression, regrowth of cartilage and/or curative treatment.Preferably disease progression is determined by the rate of cartilageloss or regrowth. In other preferred embodiments, disease progressionmay be monitored by determining the number of chondrocytes present in ajoint.

In other preferred embodiments, the treatment of osteoarthritis includesprophylactic treatment.

In certain preferred embodiments the p75NTR(NBP) is a human p75NTR(NBP).

In other preferred embodiments the p75NTR(NBP) comprises a p75NTR(NBP)connected to one or more auxiliary molecules. Preferably, the one ormore auxiliary molecules are selected from: (a) transferrin or a portionthereof; (b) albumin or a portion thereof; (c) an immunoglobulin Fc or aportion thereof; or (d) a polyethylene glycol polymer chain. In stillother preferred embodiments the p75NTR(NBP) is connected to the one ormore auxiliary molecules via one or more linkers.

In an especially preferred embodiment the p75NTR(NBP) has the amino acidsequence according to SEQ ID NO 3.

In a particularly preferred embodiment the p75NTR(NBP) binds to any ofNGF, BDNF, NT3 or NT4/5 with a binding affinity (K_(d)) of between about5 pM to about 5 nM as measured by surface plasmon resonance at 20° C.

In a preferred embodiment the p75NTR(NBP) is for separate, sequential orsimultaneous use in a combination combined with a secondpharmacologically active compound. Preferably the secondpharmacologically active compound of the combination is selected from anopioid analgesic, a nonsteroidal anti-inflammatory drug (NSAID), abarbiturate sedative, a benzodiazepine having a sedative action, an H1antagonist having a sedative action, a sedative such as glutethimide,meprobamate, methaqualone or dichloralphenazone; a skeletal musclerelaxant; an NMDA receptor antagonist, an alpha-adrenergic, a tricyclicantidepressant, an anticonvulsant, a tachykinin (NK) antagonist,particularly an NK-3, NK-2 or NK-1 antagonist, a muscarinic antagonist,a COX-2 selective inhibitor, a coal-tar analgesic, in particularparacetamol; a neuroleptic a vanilloid receptor agonist or antagonist; abeta-adrenergic; a local anaesthetic; a corticosteroid; a 5-HT receptoragonist or antagonist; a 5-HT2A receptor antagonist; a cholinergic(nicotinic) analgesic; Tramadol®; a PDEV inhibitor; a cannabinoid;metabotropic glutamate subtype 1 receptor (mGluR1) antagonist; aserotonin reuptake inhibitor; a noradrenaline (norepinephrine) reuptakeinhibitor; a dual serotonin-noradrenaline reuptake inhibitor; aninducible nitric oxide synthase (iNOS) inhibitor; anacetylcholinesterase inhibitor; a prostaglandin E2 subtype 4 (EP4)antagonist; a leukotriene B4 antagonist; a 5-lipoxygenase inhibitor; asodium channel blocker; or a 5-HT3 antagonist, and the pharmaceuticallyacceptable salts and solvates thereof.

Preferably the p75NTR(NBP) is formulated for oral, sublingual, buccal,topical, rectal, inhalation, transdermal, subcutaneous, intravenous,intra-arterial, intramuscular, intracardiac, intraosseous,intrasynovial, intradermal, intraperitoneal, transmucosal, vaginal,intravitreal, intra-articular, peri-articular, local or epicutaneousadministration.

In a further aspect of the present invention there is provided a nucleicacid encoding a p75NTR(NBP), for use in the treatment of osteoarthritisas defined above.

In another aspect of the present invention there is provided areplicable expression vector for transfecting a cell, comprising anucleic acid encoding a p75NTR(NBP), for use in the treatment ofosteoarthritis as defined above.

In yet another aspect of the present invention there is provided a hostcell expressing a p75NTR(NBP), for use in the treatment ofosteoarthritis as defined above.

In a still further aspect of the present invention there is provided apharmaceutical composition, comprising the p75NTR(NBP), the nucleic acidmolecule, the replicable expression vector, or the host cell describedabove, and a pharmaceutically acceptable carrier and/or an excipient.

Another aspect of the invention pertains to a kit comprising:

-   -   a. the p75NTR(NBP), the nucleic acid molecule, the replicable        expression vector, the host cell, or the pharmaceutical        composition described above; and    -   b. instructions for the administration of an effective amount of        the p75NTR(NBP), nucleic acid molecule, replicable expression        vector or pharmaceutical composition to an individual for any        one or more of the prevention or treatment of osteoarthritis        and/or a symptom of osteoarthritis or for ameliorating,        controlling, reducing incidence of, or delaying or reversing the        development or progression of osteoarthritis and/or a symptom of        osteoarthritis.

In another aspect of the present invention there is provided a method oftreating and/or preventing osteoarthritis and/or a symptom ofosteoarthritis in an individual comprising administering to saidindividual a therapeutically effective amount of the p75NTR(NBP), thenucleic acid molecule, the replicable expression vector, the host cell,or the pharmaceutical composition described above, optionally furthercomprising a pharmaceutically acceptable carrier.

DESCRIPTION OF FIGURES

The present invention will be further understood by reference to theattached figures, in which:

FIG. 1: Progression of loss of cartilage area following injection of MIAor ETF-PBS. Data points are mean±SEM, n=6

FIG. 2: Medial aspect of the rat knee stained with haematoxylin andeosin from animals treated with control antibody (A,B) or p75NTR 0.3mg/kg (C,D), 1 mg/kg (E,F), 3 mg/kg (G,H). Experimental osteoarthritiswas induced in the left knee (A,C,E,G) of each animal by intra-articularinjection of MIA. The right knee was injected with ETF-PBS (control;B,D,F,H). (×4 magnification) Each set of left and right knee images aretaken from an individual animal to show the contralateral control.

FIG. 3: Medial aspect of the rat knee stained with safranin O fromanimals treated with control antibody (A,B) or p75NTR 3 mg/kg (C,D).Experimental osteoarthritis was induced in the left knee (A and C) ofeach animal by intra-articular injection of MIA. The right knee wasinjected with ETF-PBS (control; B and D). (×4 magnification) Each set ofleft and right knee images are taken from an individual animal to showthe contralateral control

FIG. 4: Total cartilage area (from cartilage surface down to the borderbetween the calcified cartilage and subchondral bone) in rear kneejoints on Day 26. Significant difference compared to control antibodyare denoted thus * P<0.1 and ** P<0.05.

FIG. 5: Cartilage area stained by safranin O at threshold absorptionwavelength of 130 nm in rear knee joints following 26 days of treatment.Data are mean±SEM, n=6 Significant difference compared to controlantibody are denoted thus ** P<0.05.

FIG. 6: Amino acid sequence of a p75NTR(NBP)-Fc fusion protein (SEQ IDNo. 1). The alpha and gamma secretase cleavage sites are shown in boldtype. The IgG1 Fc portion is shown in italics.

FIG. 7: Translation product (SEQ ID No. 2), from start to stop codons,of the nucleic acid sequence set forth in FIG. 9 (SEQ ID No. 4).

FIG. 8: Amino acid sequence of a preferred p75NTR(NBP)-Fc fusion protein(SEQ ID No. 3). The IgG1 Fc portion is shown in italics. The linkersequence between the p75NTR(NBP) and Fc portions is shown underlined.

FIG. 9: Nucleic acid sequence of full product gene from 5′ cloning siteto 3′ cloning site (SEQ ID No. 4)

FIG. 10: p75-NTR(NBP)-Fc fusion protein variants: 1: p75_NTR—The p75-NTRsequence (SEQ ID No. 6); 2: Commercially available p75-NTR-Fc fusionprotein (SEQ ID No. 7); 3: p75_Fc—The commercially available p75-NTR-Fcfusion protein with the Fc sequence modified to that of the Lonzaconstant region of IgG1za (SEQ ID No. 8); 4: p75_Fc_C222S—Thecommercially available p75-NTR-Fc fusion protein with the Fc sequencemodified to that of the Lonza constant region of IgG1za and anadditional cysteine to serine mutation at position 222 (SEQ ID No. 9);5: p75_Fc_G4x1—Variant 1, a proposed p75-NTR-Fc fusion protein with afour residue glycine linker (SEQ ID No. 10); 6: p75_Fc_G4Sx1—variant 2,a proposed p75-NTR-Fc fusion protein with a single tetra-glycine serinelinker (SEQ ID No. 11); 7: p75_Fc_G4Sx2—variant 3, a proposed p75-NTR-Fcfusion protein with two tetra-glycine serine linkers (SEQ ID No. 12); 8:Lonza constant region of IgG1za (SEQ ID No. 13).

In this alignment a formatting scheme is used to highlight regions ofsimilarity between the putative receptors, the Fc-fusion protein and theFc constant region: Boxed type is used to indicate regions of identicalsequence between the variant proteins and the p75-NTR; Singleunderlining is used to indicate regions of identical sequence betweenall of the Fc-fusion proteins and the Lonza IgG1za Fc; Italics are usedto indicate linker regions at the junction of the p75-NTR and the Fcconstant region; Double-underlining and bold type are used to indicatethe position of non-identical sequence outside the linker region, at theposition equivalent to 222 in the parental p75-NTR Fc-fusion protein.

FIG. 11: SEQ ID NO. 14 Human p75NTR full amino acid sequence

FIG. 12: SEQ ID NO. 15 Human p75NTR extracellular domain includingsignal sequence

FIG. 13: SEQ ID NO. 16 Human p75NTR extracellular domain without signalsequence

FIG. 14: SEQ ID NO. 17 Human p75NTR(NBP) neurotrophin binding domain 1

FIG. 15: SEQ ID NO. 18 Human p75NTR(NBP) neurotrophin binding domain 2

FIG. 16: SEQ ID NO. 19 Human p75NTR(NBP) neurotrophin binding domain 3

FIG. 17: SEQ ID NO. 20 Human p75NTR(NBP) neurotrophin binding domain 4

FIG. 18: SEQ ID NO. 21 Human p75NTR(NBP) neurotrophin binding domain 5

FIG. 19: SEQ ID NO. 22 Human Transferrin

FIG. 20: SEQ ID NO. 23 Human Albumin

FIG. 21: SEQ ID NO. 24 Human Fc IgG1

FIG. 22: SEQ ID NO. 25 Human Fc IgG2

FIG. 23: SEQ ID NO. 26 Human Fc IgG3

FIG. 24: SEQ ID NO. 27 Human Fc IgG4

FIG. 25: SEQ ID NO. 28 Human Fc Fragment Engineered For Extended SerumHalf-Life

FIG. 26: SEQ ID NO. 29 Human Fc Fragment Engineered For Lack Of EffectorFunctions

FIG. 27: SEQ ID NO. 30 p75NTR(NBP)-Fc linker

FIG. 28: SEQ ID NO. 31 p75NTR(NBP)-Fc linker

FIG. 29: SEQ ID NO. 32 p75NTR(NBP)-Fc linker

FIG. 30: Mean body weight at day 0. Weight in grams are plotted asmean±standard deviations. Animals in Group 1 were treated with 1 mg/kgp75NTR-Fc, Group 2 with 3 mg/kg p75NTR-Fc, Group 3 with 0.3 mg/kgp75NTR-Fc, Group 4 with 3 mg/kg PG-007 and Group 5 with 3 mg/kg ControlIgG Fc.

FIG. 31: Mean body weight of rats during the eight week study describedin Example 3. Weight in grams are plotted as mean±standard deviations.Animals in Group 1 were treated with 1 mg/kg p75NTR-Fc, Group 2 with 3mg/kg p75NTR-Fc, Group 3 with 0.3 mg/kg p75NTR-Fc, Group 4 with 3 mg/kgPG-007 and Group 5 with 3 mg/kg Control IgG Fc.

FIG. 32: Spontaneous pain measurements determined with time followingtreatment with test agents. Data is shown for each animal at eachtimepoint (from day 35 to day 56). Animals in Group 1 were treated with1 mg/kg p75NTR-Fc, Group 2 with 3 mg/kg p75NTR-Fc, Group 3 with 0.3mg/kg p75NTR-Fc, Group 4 with 3 mg/kg PG-007, Group 5 with 3 mg/kgControl IgG Fc, Group 6 with ETF-PBS and Group 7 were naïve. *p<0.05 and**<0.01 for one sample t-test against the theoretical mean of 0.5.

FIG. 33: Spontaneous pain measurements determined with time followingtreatment with test agents. Data is shown for each animal at eachtimepoint (from day 35 to day 56). Animals in Group 1 were treated with1 mg/kg p75NTR-Fc, Group 2 with 3 mg/kg p75NTR-Fc, Group 3 with 0.3mg/kg p75NTR-Fc, Group 4 with 3 mg/kg PG-007, Group 5 with 3 mg/kgControl IgG Fc, Group 6 with ETF-PBS and Group 7 were naïve. *p<0.05 and**<0.01 for one sample t-test against the theoretical mean of 0.5.

FIG. 34: Medial aspect of the rat knee stained with Safranin O FastGreen from animals treated with 3 mg/kg control IgG-Fc from day 28 today 56 following a low MIA or ETF-PBS injection on day 0 into the knee(×4 magnification). Representative images are taken from animals treatedwith 3 mg/kg control IgG-Fc. Each set of left and right knee image aretaken from one individual animal to show the contralateral control.

FIG. 35: Medial aspect of the rat knee stained with Safranin O FastGreen from animals treated with 3 mg/kg PG-007 from day 28 to day 56following a low MIA or ETF-PBS injection on day 0 into the knee (×4magnification). Representative images are taken from animals treatedwith 3 mg/kg PG-007. Each set of left and right knee image are takenfrom one individual animal to show the contralateral control.

FIG. 36: Medial aspect of the rat knee stained with Safranin O FastGreen from animals treated with 3 mg/kg p75NTR-Fc from day 28 to day 56following a low MIA or ETF-PBS injection on day 0 into the knee (×4magnification). Representative images are taken from animals treatedwith 3 mg/kg p75NTR-Fc. Each set of left and right knee image are takenfrom one individual animal to show the contralateral control.

FIG. 37: Medial aspect of the rat knee stained with Safranin O FastGreen from animals treated with 1 mg/kg p75NTR-Fc from day 28 to day 56following a low MIA or ETF-PBS injection on day 0 into the knee (×4magnification). Representative images are taken from animals treatedwith 1 mg/p75NTR-Fc. Each set of left and right knee image are takenfrom one individual animal to show the contralateral control.

FIG. 38: Medial aspect of the rat knee stained with Safranin O FastGreen from animals treated with 0.3 mg/kg p75NTR-Fc from day 28 to day56 following a low MIA or ETF-PBS injection on day 0 into the knee (×4magnification). Representative images are taken from animals treatedwith 0.3 mg/kg p75NTR-Fc. Each set of left and right knee image aretaken from one individual animal to show the contralateral control.

FIG. 39: Cartilage pathology grades. Administration of PG007 showed nohistologically significant efficacy effects. P75NTR-Fc showed markedefficacy on chondroprotection at 0.3 and 1.0 mg/kg. The efficacy profileat 3.0 mg/kg was more variable—with 50% of the group showing overlaywith the control group.

FIG. 40: Sub-chondral bone grades. Administration of PG007 wasassociated with histologically significant increase in sub-chondral bonepathology compared to the control group—similar to the profile observedwith 3.0 mg/kg P75NTRFc. By contrast, administration of P75NTR-Fc at 0.3and 1.0 mg/kg was associated with marked improvement in sub-chondralbone histology.

FIG. 41: Stromal cavity grades. Administration of PG007 was notassociated with any histologically significant efficacy effects. Bycontrast, administration of P75NTR-Fc at all does was associated withreduction in osteolytic pathology and expansion of stromal cavities,although the effects at 0.3 and 1.0 mg/kg were most marked—both groupsreducing pathology to almost normal levels.

FIG. 42: Cancellous bone grades. Administration of PG007 was associatedwith cancellous bone pathology similar to controls—with two samplesexceeding control ranges. By contrast, P75NTR-Fc markedly reducedcancellous bone pathology at the 0.3 and 1.0 mg/kg dose levels.P75NTF-Fc at 3.0 mg/kg presented a more variable profile, with themajority of samples showing overlay with the control group.

FIG. 43: Bone marrow hyper-cellularity. Administration of PG007 wasassociated with expansion of myeloid cells in the bone marrow, with foursamples either at the top or exceeding the control range. P75NTR-Fcreduced marrow cellularity—with reduced myeloid expansion being aprominent feature—at the 0.3 and 1.0 mg/kg doses. The 3.0 mg/kg dose,although showing a trend towards inhibition, showed overlay with lowlevel responders in the control group.

FIG. 44: Multi-focal bone sclerosis. There were no histologicallysignificant differences between the study groups—although the P75NTR-Fcsamples did show a clustering towards the top end of the control range.

FIG. 45: Osteoblast hyperplasia. Administration of P75NTR-Fc at 0.3 and1.0 mg/kg was associated with histologically significant osteoblastproliferation—with palisading and osteoblastic plate formation beingespecially prominent in the epiphyseal zone, at the sub-chondralbone/cartilage zone. In addition, although not graded, there wereprominent ‘fibroblast-like’ cells in this zone. PG007 and P75NTR-Fc (3.0mg/kg) were histologically similar to controls (FIG. 45).

DETAILED DESCRIPTION

Osteoarthritis symptoms include inflammation, pain and swelling. Inaddition, the loss of cartilage can lead to the formation of boneoutgrowths (osteophytes) which exacerbate symptoms and can lead tonarrowing and distortion of the joint. Treatment options are currentlylimited to disease management options including physiotherapy,administration of pain killers and/or administration ofanti-inflammatory drugs and, in some cases, joint replacement viasurgery. Until now, no curative treatment options for osteoarthritishave been reported. There are also no reported treatments which haltprogression of this degenerative disease. Accordingly, the presentinvention seeks to address the need for a curative osteoarthritistreatment, or at least to provide a treatment capable of haltingdegeneration in osteoarthritis.

Surprisingly, the present inventors have discovered that administrationof a p75NTR neurotrophin binding protein (p75NTR(NBP)) in an acceptedanimal model of osteoarthritis not only halted disease progression, butalso resulted in significant reversal of damage attributable toosteoarthritis progression.

Accordingly, there is provided in a first aspect a p75NTR neurotrophinbinding protein (p75NTR(NBP)) for use in the treatment ofosteoarthritis.

In preferred embodiments, the treatment of osteoarthritis includesrelief from the symptoms of osteoarthritis. Preferably relief from thesymptoms of osteoarthritis include, but are not limited to reduction inpain, inflammation, swelling, tenderness, joint stiffness or increase injoint mobility or any combination of these.

In a particularly preferred embodiment, treatment of osteoarthritisincludes slowing or arresting of disease progression and/or reduction incartilage loss. Preferably treatment of osteoarthritis includes reversalof disease progression, regrowth of cartilage and/or curative treatment.Preferably disease progression is determined by the rate of cartilageloss or regrowth. The rate of cartilage loss may be monitored by avariety of methods, including but not limited to Magnetic ResonanceImaging (MRI) or X-ray computed tomography (x-ray CT). In otherpreferred embodiments, disease progression may be monitored bydetermining the number of chondrocytes present in a joint.

The term “curative treatment” as used herein is intended to encompasstreatments which restore a patient to their pre-disease state. Suchtreatments may require continued administration of the active compoundin order to maintain a pre-disease state. Alternatively, curativetreatments may be halted once a pre-disease state is reached.

In other preferred embodiments, the treatment is of either primary orsecondary osteoarthritis. Treatment of primary osteoarthritis includestreatment of both primary generalized nodal osteoarthritis and erosiveosteoarthritis (EOA, also called inflammatory osteoarthritis).

Treatment of osteoarthritis is also intended to include amelioration ofdisease symptoms as classified under the WOMAC grading or Outerbridgeclassification systems. In preferred embodiments, the treatment ofosteoarthritis results in reversal of disease progression, leading to achange of disease stage (WOMAC grading) or grade (Outerbridgeclassification).

In other preferred embodiments, the treatment of osteoarthritis includesprophylactic treatment.

In certain preferred embodiments the p75NTR(NBP) is a human p75NTR(NBP).

In other preferred embodiments the p75NTR(NBP) comprises a p75NTR(NBP)connected to one or more auxiliary molecules. Preferably, the one ormore auxiliary molecules are selected from: (a) transferrin or a portionthereof; (b) albumin or a portion thereof; (c) an immunoglobulin Fc or aportion thereof; or (d) a polyethylene glycol polymer chain. In stillother preferred embodiments the p75NTR(NBP) is connected to the one ormore auxiliary molecules via one or more linkers. Preferably the linkeris selected from: (a) a covalent bond; (b) a non-covalent bond; (c) apeptide bond; or (d) one amino acid or a plurality of amino acidscomprising a peptide. In a particularly preferred embodiment, thep75NTR(NBP) comprises a p75NTR(NBP) connected to an immunoglobulin Fc ora portion thereof, optionally via a linker.

Where the p75NTR(NBP) is connected to more than one auxiliary molecule,optionally each auxiliary molecule is either the same or different or amixture of the same and different. Similarly, where the p75NTR(NBP) isconnected to more than one auxiliary molecule via one or more linkers,optionally each linker is either the same or different or a mixture ofthe same and different

In an especially preferred embodiment the p75NTR(NBP) has the amino acidsequence according to SEQ ID NO 3.

In a particularly preferred embodiment the p75NTR(NBP) binds to any ofNGF, BDNF, NT3 or NT4/5 with a binding affinity (K_(d)) of between about5 pM to about 5 nM as measured by surface plasmon resonance at 20° C.

In a preferred embodiment the p75NTR(NBP) is for separate, sequential orsimultaneous use in a combination combined with a secondpharmacologically active compound. Preferably the secondpharmacologically active compound of the combination is selected from anopioid analgesic, a nonsteroidal anti-inflammatory drug (NSAID), abarbiturate sedative, a benzodiazepine having a sedative action, an H1antagonist having a sedative action, a sedative such as glutethimide,meprobamate, methaqualone or dichloralphenazone; a skeletal musclerelaxant; an NMDA receptor antagonist, an alpha-adrenergic, a tricyclicantidepressant, an anticonvulsant, a tachykinin (NK) antagonist,particularly an NK-3, NK-2 or NK-1 antagonist, a muscarinic antagonist,a COX-2 selective inhibitor, a coal-tar analgesic, in particularparacetamol; a neuroleptic a vanilloid receptor agonist or antagonist; abeta-adrenergic; a local anaesthetic; a corticosteroid; a 5-HT receptoragonist or antagonist; a 5-HT2A receptor antagonist; a cholinergic(nicotinic) analgesic; Tramadol®; a PDEV inhibitor; a cannabinoid;metabotropic glutamate subtype 1 receptor (mGluR1) antagonist; aserotonin reuptake inhibitor; a noradrenaline (norepinephrine) reuptakeinhibitor; a dual serotonin-noradrenaline reuptake inhibitor; aninducible nitric oxide synthase (iNOS) inhibitor; anacetylcholinesterase inhibitor; a prostaglandin E2 subtype 4 (EP4)antagonist; a leukotriene B4 antagonist; a 5-lipoxygenase inhibitor; asodium channel blocker; or a 5-HT3 antagonist, and the pharmaceuticallyacceptable salts and solvates thereof.

Preferred opioid analgesics include, but are not limited to, morphine,heroin, hydromorphone, oxymorphone, levorphanol, levallorphan,methadone, meperidine, fentanyl, cocaine, codeine, dihydrocodeine,oxycodone, hydrocodone, propoxyphene, nalmefene, nalorphine, naloxone,naltrexone, buprenorphine, butorphanol, nalbuphine or pentazocine.

Preferred nonsteroidal anti-inflammatory drugs (NSAIDs) include, but arenot limited to, aspirin, diclofenac, diflusinal, etodolac, fenbufen,fenoprofen, flufenisal, flurbiprofen, ibuprofen, indomethacin,ketoprofen, ketorolac, meclofenamic acid, mefenamic acid, meloxicam,nabumetone, naproxen, nimesulide, nitroflurbiprofen, olsalazine,oxaprozin, phenylbutazone, piroxicam, sulfasalazine, sulindac, tolmetinor zomepirac.

Preferred barbiturate sedatives include, but are not limited to,amobarbital, aprobarbital, butabarbital, butabital, mephobarbital,metharbital, methohexital, pentobarbital, phenobartital, secobarbital,talbutal, theamylal or thiopental.

Preferred benzodiazepines having a sedative action include, but are notlimited to, chlordiazepoxide, clorazepate, diazepam, flurazepam,lorazepam, oxazepam, temazepam or triazolam.

Preferred H1 antagonists having a sedative action include, but are notlimited to, diphenhydramine, pyrilamine, promethazine, chlorpheniramineor chlorcyclizine.

Preferred sedatives include, but are not limited to, glutethimide,meprobamate, methaqualone or dichloralphenazone.

Preferred skeletal muscle relaxants include, but are not limited to,baclofen, carisoprodol, chlorzoxazone, cyclobenzaprine, methocarbamol ororphrenadine.

Preferred NMDA receptor antagonists include, but are not limited to,dextromethorphan ((+)-3-hydroxy-N-methylmorphinan) or its metabolitedextrorphan ((+)-3-hydroxy-N-methylmorphinan), ketamine, memantine,pyrroloquinoline quinine, cis-4-(phosphonomethyl)-2-piperidinecarboxylicacid, budipine, EN-3231 (MorphiDex®, a combination formulation ofmorphine and dextromethorphan), topiramate, neramexane or perzinfotelincluding an NR2B antagonist, e.g. ifenprodil, traxoprodil or(−)-(R)-6-{2-[4-(3-fluorophenyl)-4-hydroxy-1-piperidinyl]-1-hydroxyethyl-3,4-dihydro-2(1H)-quinolinone.

Preferred alpha-adrenergics include, but are not limited to, doxazosin,tamsulosin, clonidine, guanfacine, dexmetatomidine, modafinil, or4-amino-6,7-dimethoxy-2-(5-methane-sulfonamido-1,2,3,4-tetrahydroisoquinol-2-yl)-5-(2-pyridyl)quinazoline.

Preferred tricyclic antidepressants include, but are not limited to,desipramine, imipramine, amitriptyline or nortriptyline.

Preferred anticonvulsants include, but are not limited to,carbamazepine, lamotrigine, topiratmate or valproate.

Preferred tachykinin (NK) antagonists include, but are not limited to,(alphaR,9R)-7-[3,5-bis(trifluoromethyl)benzyl]-8,9,10,11-tetrahydro-9-methyl-5-(4-methylphenyl)-7H-[1,4]diazocino[2,1-g][1,7]-naphthyridine-6-13-dione(TAK-637),5-[[(2R,3S)-2-[(1R)-1-[3,5-bis(trifluoromethyl)phenyl]ethoxy-3-(4-fluorophenyl)-4-morpholinyl]-methyl]-1,2-dihydro-3H-1,2,4-triazol-3-one(MK-869), aprepitant, lanepitant, dapitant or3-[[2-methoxy-5-(trifluoromethoxy)phenyl]-methylamino]-2-phenylpiperidine(2S,3S).

Preferred muscarinic antagonists include, but are not limited to,oxybutynin, tolterodine, propiverine, tropsium chloride, darifenacin,solifenacin, temiverine and ipratropium. Preferred COX-2 selectiveinhibitors include, but are not limited to, celecoxib, rofecoxib,parecoxib, valdecoxib, deracoxib, etoricoxib, or lumiracoxib.

Preferred coal-tar analgesics include, but are not limited to,paracetamol.

Preferred neuroleptics include, but are not limited to, droperidol,chlorpromazine, haloperidol, perphenazine, thioridazine, mesoridazine,trifluoperazine, fluphenazine, clozapine, olanzapine, risperidone,ziprasidone, quetiapine, sertindole, aripiprazole, sonepiprazole,blonanserin, iloperidone, perospirone, raclopride, zotepine, bifeprunox,asenapine, lurasidone, amisulpride, balaperidone, palindore,eplivanserin, osanetant, rimonabant, meclinertant, Miraxion® orsarizotan.

Preferred vanilloid receptor agonists include, but are not limited to,resinferatoxin. Preferred vanilloid receptor antagonists include, butare not limited to, capsazepine.

Preferred beta-adrenergics include, but are not limited to, propranolol.Preferred local anaesthetics include, but are not limited to,mexiletine. Preferred corticosteroids include, but are not limited to,dexamethasone.

Preferred 5-HT receptor agonists or antagonists, particularly 5-HT1B/1Dagonists include, but are not limited to, eletriptan, sumatriptan,naratriptan, zolmitriptan or rizatriptan; Preferred 5-HT2A receptorantagonists include, but are not limited to,R(+)-alpha-(2,3-dimethoxy-phenyl)-1-[2-(4-fluorophenylethyl)]-4-piperidinemethanol(MDL-100907).

Preferred cholinergic (nicotinic) analgesics include, but are notlimited to, ispronicline (TC-1734),(E)-N-methyl-4-(3-pyridinyl)-3-buten-1-amine (RJR-2403),(R)-5-(2-azetidinylmethoxy)-2-chloropyridine (ABT-594) or nicotine.

Preferred PDEV inhibitors include, but are not limited to,5-[2-ethoxy-5-(4-methyl-1-piperazinyl-sulphonyl)phenyl]-1-methyl-3-n-propyl-1,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-one(sildenafil),(6R,12aR)-2,3,6,7,12,12a-hexahydro-2-methyl-6-(3,4-methylenedioxyphenyl)-pyrazino[2′,1′:6,1]-pyrido[3,4-b]indole-1,4-dione(IC-351 or tadalafil),2-[2-ethoxy-5-(4-ethyl-piperazin-1-yl-1-sulphonyl)-phenyl]-5-methyl-7-propyl-3H-imidazo[5,1-f][1,2,4]triazin-4-one(vardenafil),5-(5-acetyl-2-butoxy-3-pyridinyl)-3-ethyl-2-(1-ethyl-3-azetidinyl)-2,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-one,5-(5-acetyl-2-propoxy-3-pyridinyl)-3-ethyl-2-(1-isopropyl-3-azetidinyl)-2,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-one,5-[2-ethoxy-5-(4-ethylpiperazin-1-ylsulphonyl)pyridin-3-yl]-3-ethyl-2-[2-methoxyethyl]-2,6-dihydro-7H-pyrazolo[4,3-d]pyrimidin-7-one,4-[(3-chloro-4-methoxybenzyl)amino]-2-[(2S)-2-(hydroxymethyl)pyrrolidin-1-yl]-N-(pyrimidin-2-ylmethyl)pyrimidine-5-carboxamide,3-(1-methyl-7-oxo-3-propyl-6,7-dihydro-1H-pyrazolo[4,3-d]pyrimidin-5-yl)-N-[2-(1-methylpyrrolidin-2-yl)ethyl]-4-propoxybenzenesulfonamide.

Preferred cannabinoids include, but are not limited to,tetrahydrocannabinol, cannabinol, cannabidiol, cannabigerol,tetrahydrocannabivarin, cannabidivarin and cannabichromene.

Preferred serotonin reuptake inhibitors include, but are not limited to,sertraline, sertraline metabolite demethylsertraline, fluoxetine,norfluoxetine (fluoxetine desmethyl metabolite), fluvoxamine,paroxetine, citalopram, citalopram metabolite desmethylcitalopram,escitalopram, d,l-fenfluramine, femoxetine, ifoxetine, cyanodothiepin,litoxetine, dapoxetine, nefazodone, cericlamine and trazodone.

Preferred noradrenaline (norepinephrine) reuptake inhibitors include,but are not limited to, maprotiline, lofepramine, mirtazepine,oxaprotiline, fezolamine, tomoxetine, mianserin, buproprion, buproprionmetabolite hydroxybuproprion, nomifensine and viloxazine (Vivalan®),especially a selective noradrenaline reuptake inhibitor such asreboxetine, in particular (S,S)-reboxetine.

Preferred dual serotonin-noradrenaline reuptake inhibitors include, butare not limited to, venlafaxine, venlafaxine metaboliteO-desmethylvenlafaxine, clomipramine, clomipramine metabolitedesmethylclomipramine, duloxetine, milnacipran and imipramine.

Preferred inducible nitric oxide synthase (iNOS) inhibitors include, butare not limited to, S-[2-[(1-iminoethyl)amino]ethyl]-L-homocysteine,S-[2-[(1-iminoethyl)-amino]ethyl]-4,4-dioxo-L-cysteine,S-[2-[(1-iminoethyl)amino]ethyl]-2-methyl-L-cysteine,(2S,5Z)-2-amino-2-methyl-7-[(1-iminoethyl)amino]-5-heptenoic acid,2-[[(1R,3S)-3-amino-4-hydroxy-1-(5-thiazolyl)-butyl]thio]-5-chloro-3-pyridinecarbonitrile;2-[[(1R,3S)-3-amino-4-hydroxy-1-(5-thiazolyl)butyl]thio]-4-chlorobenzonitrile,(2S,4R)-2-amino-4-[[2-chloro-5-(trifluoromethyl)phenyl]thio]-5-thiazolebutanol,2-[[(1R,3S)-3-amino-4-hydroxy-1-(5-thiazolyl)butyl]thio]-6-(trifluoromethyl)-3 pyridinecarbonitrile,2-[[(1R,3S)-3-amino-4-hydroxy-1-(5-thiazolyl)butyl]thio]-5-chlorobenzonitrile,N-[4-[2-(3-chlorobenzylamino)ethyl]phenyl]thiophene-2-carboxamidine, orguanidinoethyldisulfide.

Preferred acetylcholinesterase inhibitors include, but are not limitedto, donepezil. Preferred prostaglandin E2 subtype 4 (EP4) antagonistsinclude, but are not limited to,N-[({2-[4-(2-ethyl-4,6-dimethyl-1H-imidazo[4,5-c]pyridin-1-yl)phenyl]ethyl}amino)-carbonyl]-4-methylbenzenesulfonamideor4-[(1S)-1-({[5-chloro-2-(3-fluorophenoxy)pyridin-3-yl]carbonyl}amino)ethyl]benzoicacid.

Preferred leukotriene B4 antagonists include, but are not limited to,1-(3-biphenyl-4-ylmethyl-4-hydroxy-chroman-7-yl)-cyclopentanecarboxylicacid (CP-105696),5-[2-(2-Carboxyethyl)-3-[6-(4-methoxyphenyl)-5E-hexenyl]oxyphenoxy]-valericacid (ONO-4057) or DPC-11870.

Preferred 5-lipoxygenase inhibitors include, but are not limited to,zileuton,6-[(3-fluoro-5-[4-methoxy-3,4,5,6-tetrahydro-2H-pyran-4-yl])phenoxy-methyl]-1-methyl-2-quinolone(ZD-2138), or 2,3,5-trimethyl-6-(3-pyridylmethyl),1,4-benzoquinone(CV-6504).

Preferred sodium channel blockers include, but are not limited to,lidocaine. Preferred 5-HT3 antagonists include, but are not limited to,ondansetron.

Preferably the p75NTR(NBP) is formulated for oral, sublingual, buccal,topical, rectal, inhalation, transdermal, subcutaneous, intravenous,intra-arterial, intramuscular, intracardiac, intraosseous,intrasynovial, intradermal, intraperitoneal, transmucosal, vaginal,intravitreal, intra-articular, peri-articular, local or epicutaneousadministration.

In a further aspect of the present invention there is provided a nucleicacid encoding a p75NTR(NBP), for use in the treatment of osteoarthritisas defined above.

In another aspect of the present invention there is provided areplicable expression vector for transfecting a cell, comprising anucleic acid encoding a p75NTR(NBP), for use in the treatment ofosteoarthritis as defined above.

In yet another aspect of the present invention there is provided a hostcell expressing a p75NTR(NBP), for use in the treatment ofosteoarthritis as defined above.

In a still further aspect of the present invention there is provided apharmaceutical composition, comprising the p75NTR(NBP), the nucleic acidmolecule, the replicable expression vector, or the host cell describedabove, and a pharmaceutically acceptable carrier and/or an excipient.

Preferred pharmaceutically acceptable carriers include, but are notlimited to, [ . . . ]

Another aspect of the invention pertains to a kit comprising:

-   -   a. the p75NTR(NBP), the nucleic acid molecule, the replicable        expression vector, the host cell, or the pharmaceutical        composition described above; and    -   b. instructions for the administration of an effective amount of        the p75NTR(NBP), nucleic acid molecule, replicable expression        vector or pharmaceutical composition to an individual for any        one or more of the prevention or treatment of osteoarthritis        and/or a symptom of osteoarthritis or for ameliorating,        controlling, reducing incidence of, or delaying or reversing the        development or progression of osteoarthritis and/or a symptom of        osteoarthritis.

In another aspect of the present invention there is provided a method oftreating and/or preventing osteoarthritis and/or a symptom ofosteoarthritis in an individual comprising administering to saidindividual a therapeutically effective amount of the p75NTR(NBP), thenucleic acid molecule, the replicable expression vector, the host cell,or the pharmaceutical composition described above, optionally furthercomprising a pharmaceutically acceptable carrier.

Examples Example 1—Affinity Measurements of p75NTR Using Biacore

Methods

The kinetics and affinity of p75NTR are determined by surface plasmonresonance technology using a Biacore T200 (GE Healthcare, Sweden). TheBiacore methods are based on those recommended by Abdiche andcolleagues. (Abdiche, et al., 2008) Protein A (10 μg/mL in 10 mM sodiumacetate buffer) is immobilised on the surface of a CM5 biosensor chip bythe amine coupling method using 1-ethyl-3-(3-dimethylaminopropylcarbodiimide) (EDC) and N-hydroxy-succinimide (NHS) and ethanolamine asprovided in the amine coupling kit.

Briefly, the steps involved in the amine-coupling immobilisation wizardon the Biacore instrument are:

-   -   EDC and NHS are mixed 1:1    -   EDC/NHS mixture is injected over each flow cell of a CM5 chip        for 420 seconds at 10 μL/min    -   Protein A [10 μg/mL] in 10 mM sodium acetate buffer, pH 4.5 is        injected over the same flow cell for 420 seconds at 10 μL/min    -   Ethanolamine is injected over the same flow cell for 420 seconds        at 10 μL/min

Approximately 2200-2900 response units (RU) are immobilised. One flowcell is set as the blank control. p75NTR-Fc is captured onto the otherflow cell of the biosensor chip at 15° C. using a 30 second injection ofp75NTR (10 μg/mL in HBS-EP [0.01 M HEPES pH 7.4, 0.15 M NaCl, 3 mM EDTA,0.005% v/v Surfactant P20]) at a flow rate of 10 μL/min to achieve thedesired p75NTR RU level (˜400 RU; calculated using the molecular weightsof the p75NTR, the relevant neurotrophin and stoichiometric ratio).Immobilising p75NTR rather than the ligand helps ensure theneurotrophins are in their native states.

A single-cycle kinetics study is performed by injecting increasingconcentrations of neurotrophin in HBS-EP buffer over the flow cells for120 seconds at 30 μL/min per neurotrophin concentration. Following thefinal neurotrophin injection HBS-EP is flowed over the chip for 600seconds to determine dissociation rates. Chip sensor surfaces areregenerated back to their Protein A surface by injecting 10 mM GlycineHCl, pH 2 for 60 seconds at 30 μL/min prior to every new injectioncycle.

Multi-cycle kinetics studies are performed by injecting the lowestneurotrophin oncentration over the flow cells for 300 seconds at 30μL/min, followed by HBS-EP buffer for 300 seconds at 30 μL/min. The chipis then regenerated by 2×60 second injections of 10 mM Glycine, HCl pH 2and the cycle repeated for each increasing neurotrophin concentration.

Results

p75NTR binds reversibly to the neurotrophins NGF, BDNF, NT-3 and NT-4.The affinities measured using Biacore are given in Table 1.

TABLE 1 Affinity of p75NTR for the four neurotrophins NeurotrophinAffinity (pM) Affinity Range (pM) NGF 554 50-5000 BDNF 41.8 5-500 NT-314.2 1-100 NT-4 181 10-1000

Example 2—Monoiodoacetate-Induced Osteoarthritis Study

Experimental osteoarthritis induced by intra-articular injection ofmonoiodoactetate (MIA) in the rear knee of rats is a well-recognisedmodel of osteoarthritis. The pathological progression of the disease andpain behaviour have been reported (Guzman, et al., 2003) (Fernihough, etal., 2004). MIA disrupts glycolysis by inhibition ofglyceraldehyde-3-phosphate dehydrogenase, leading to chondrocyte death(Harvey & Dickenson, 2009). The structural integrity of cartilage relieson the normal functioning of chondrocytes, thus MIA-induced loss ofchondrocytes leads to cartilage degeneration and changes of thesubchondral bone consistent with the clinical histopathology of OA(Janusz, et al., 2001; Kobayashi, et al., 2003; Naveen, et al., 2014).Injection of 0.3 mg MIA induces loss of cartilage over a 10-week period.Cartilage loss is greatest between Week 3 and Week 6 (FIG. 1).

44 male Wistar rats (Charles River, UK) housed in pairs and weighing200-250 g on Day −2, are randomly assigned by pair to treatment suchthat the mean body weight of each treatment group is similar. On Day 0the rats receive treatment as indicated in Table 1. Freshly preparedhuman IgG (3.25 mg/mL in endotoxin free phosphate buffered saline[ETF-PBS]) and p75NTR (3.25 mg/mL in EF-PBS) are administeredsubcutaneously. The laboratory personnel are blinded to the treatment ofthe animals throughout the study.

TABLE 2 Treatment groups. MIA, monoiodiacetate; IgG, immunoglobulin GTreatment group (n) MIA Antibody 1 (6) 0.3 mg Human IgG 3.0 mg/kg 2 (6)0.3 mg p75NTR 0.3 mg/kg 3 (6) 0.3 mg p75NTR 1.0 mg/kg 4 (6) 0.3 mgp75NTR 3.0 mg/kg

Three hours later all animals are anaesthetised with isofluorane. Eachanaesthetised animal receives an intra-articular injection of 50 μLEF-PBS containing 0.3 mg MIA in either the right or left knee, accordingto the randomisation schedule. The contralateral knee of eachanaesthetised animal is injected with 50 μL EF-PBS. A further respectiveantibody or p75NTR treatment is administered on Day 5 and 15.

The study terminates on Day 26, when cartilage loss is evident andactive (FIG. 1). Animals are anaesthetised using isoflourane, terminalblood samples are taken by cardiac puncture and plasma samples prepared.The skin on the lower hind legs is removed and the muscle bundlesseparated from the bones but left intact with the knee. The femur,fibula and tibia are severed and the knee with attached muscle is placedin 10% neutral buffered formalin. Tissue samples are kept in bufferedformalin for 48-72 hours before processing for histological analysis.

Tissue samples are prepared for light microscopy using standardprocedures as soon as possible after collection to minimise damagecaused from the formalin fixation. The samples are decalcified in 8%formic acid for 10 days, processed using a Shandon Citadel tissueprocesser and embedded into molten paraffin wax. At least four sections(10 μm) from each rat knee tissue block are processed for standardHaematoxylin and Eosin (H&E) staining using an automated linear stainingmachine (Leica ST4040). Further sections are stained using Safranin O.Slides are viewed at either times four or ten magnification and imageanalysis is performed using a computerised system. The total cartilagearea, from cartilage surface down to the border between the calcifiedcartilage and subchondral bone is measured. Light absorption of stainbound to tissue sections is quantified under monochromatic light withdigital densitometry. The intensity of the red staining of theglycosaminoglycans by Safranin O is quantitated by measuring thecartilage area stained when the absorption threshold is set at 130 nm.

The concentration of control antibody and exogenous p75NTR in plasma isestimated by measuring human IgG using an enzyme-linked immunosorbentassay (ELISA).

Results—Effect of p75NTR on Histological Changes in the Knee FollowingMIA-Induced OA

No tissue degeneration nor features of OA were observed in knees thathad been injected with ETF-PBS in animals from all treatment groups(FIG. 2B,D,F,H, FIG. 3B,D,F,H). In contrast, MIA-injected knees inanimals treated with control antibody showed areas of mild chondrocytedegeneration, which frequently involved the entire thickness of thearticular cartilage (FIG. 2A). There was evidence of a recentinflammatory response in some areas, marked by leukocyte accumulation.Treatment with p75NTR did not accelerate the progression of OA (FIG.2C,E,G). There were fewer histological changes in the architecture oflow MIA treated knees treated with p75NTR compared with animals treatedwith control antibodies, indicating protection from injury or repair ofdamage. This was evident at all p75NTR concentrations tested (0.3-3.0mg/kg). Indeed, animals treated with the highest dose of p75NTR-Fc hadsignificant (P<0.05) increase in cartilage area (evidence of p75NTR-Fcinduced repair in OA see FIG. 4).

Effect of p75NTR on Proteoglycan Density in the Knee FollowingMIA-Induced OA

The overall cartilage area in MIA-treated knees was improved in animalstreated with p75NTR-Fc: this was significantly different (P<0.05) foranimals treated with p75NTR-Fc at 3.0 mg/kg compared to correspondingcontrol animals on Day 26 post intra-articular injection (FIG. 4).

The intensity of safranin O staining, as assessed by image analysis,which is proportional to the glycosaminoglycan content of cartilage,showed marked differences (P<0.05) between the treatment groups (seeFIG. 5). Using an absorption threshold of 130 nm, only 20% of thecartilage from MIA-treated knee was red-stained above the 130 nmthreshold in animals treated with control antibodies compared with thecontralateral knees, indicating a substantial loss ofglycosaminoglycans. In contrast, approximately 50% of the cartilage inthe MIA-treated knees of animals receiving p75NTR-Fc was above the 130nm threshold (FIG. 5). This indicates that p75NTR-Fc is preventing theongoing loss of structural integrity of the cartilage following MIAinjection and moreover disease modification in OA.

Example 3—Effects of p75NTR-Fc on Regression of Osteoarthritis in RatCompared to PG-007

The aim of this study is to assess whether ascending doses of p75NTR-Fccan cause regression of OA once the disease has been established. Inaddition the efficacy of the treatment in terms of the pain associatedwith MIA induced arthritis in the rat has been assessed and comparedwith treatment with the Tanezumab like anti-Neurotrophin Growth Factor(NGF) antibody, PG-007.

Protocol

Reagents

TABLE 3 Details of reagents used in Example 3 Source and catalogueReagent number Batch number Monosodium Iodoacetate Sigma I2512#SLBB6147V (MIA) Endotoxin free PBS (ETF- Promocell C-40240 359P109 PBS)10% neutral buffered Sigma HT501320 090M4370 formalin solution EDTA(dipotassium salt Sigma ED2P BCBF9021V dihydrate)

Preparation of MIA

MIA was prepared at a concentration of 0.3 mg/50 μl ETF-PBS (the volumeused for each intra-articular injection) which is equivalent to 6 mg/mlMIA stock solution. 68 mg of MIA was weighed out and dissolved in 11.3ml ETF-PBS. The MIA was prepared a day in advance and was stored at 4°C. in the dark until required. All animals received the MIA preparedfrom the same batch.

Test Agents

TABLE 4 Details of test agents used in Example 3 Source and catalogueTest agent number Batch number PG-007 Lonza at 3.25 mg/ml, 0.026 EU/mgBatch 1 (provided by Levicept) p75NTR-Fc 2.90 mg/ml stock Human IgG Fcfragment AbCam AB90285 prepared GR196868-1 full-length protein at 2.15mg/ml in ETF-PBS

Preparation of Test Agents

The test agents used in this study were prepared fresh on the specificday that the animals were dosed (see Table 4). Wherever possible thevolumes of each antibody prepared were equal so that the in vivoscientist injecting the antibody was unaware of what treatment was ineach vial (see Table 5).

TABLE 5 Typical preparation of test agents used in Example 3 Stock StockVehicle Final Final Dose Concen- vol- vol- vol- concen- vol- Dosetration ume ume ume tration ume Antibody (mg/kg) (mg/ml) (ml) (ml) (ml)(mg/ml) (ml/kg) PG-007 3 3.25 3.30 14.70 18 0.60 5 p75NTR- 3 2.90 3.7314.27 18 0.60 5 Fc p75NTR- 1 2.90 1.24 16.76 18 0.20 5 Fc p75NTR- 0.32.90 0.37 17.63 18 0.06 5 Fc IgG-Fc 3 2.15 5.00 13.00 18 0.60 5

Animals

39 male Wistar rats (from Charles River, UK) weighing 120-150 g onarrival were used in Example 3. Each animal was checked on arrival andappeared outwardly healthy. They were randomly assigned to a cage ofthree and each rat was allocated a unique identification number by atattoo on the tail. Animals were acclimatised to the animal unit for atleast ten days prior to the start of the study on day 0.

Once the rats had acclimatised to their environment they weretransferred to a stock/procedure room, where all the in vivo procedureswere carried out. Animals were kept illuminated by fluorescent lightsset to give a 12 hour light-dark cycle (on 07.00 off 19.00) asrecommended in the Home Office Animals (Scientific Procedures) Act 1986.The rooms were air-conditioned and the air temperature (21° C.+/−2° C.)and relative humidity were routinely measured.

Rats were fed R105-25 irradiated complete diet for rats (ScientificAnimal Food and Engineering, Augy, France) and autoclaved water wasavailable ad libitum. Each batch of diet was checked and screenedroutinely for composition and contaminants. Nesting and cages wereautoclaved and each cage was individually ventilated (IVC system).

Experimental Design

Following the induction of arthritis by the intraarticular injection ofMIA in one knee and once were significant differences were observed inthe weight bearing (incapacitance measurements) of the limbs treatedwith MIA compared to its contralateral control limb treated withETF-PBS, animals were randomised into five treatment groups ofequivalent nociceptive behaviour (n=6 animals per group) and one vehiclecontrol group (n=3). Six naïve animals were included as negativecontrols.

TABLE 6 Overview of experimental design for Example 3 Intra-articularTreatment (group) injection in one by the subcutaneous Terminal knee (n)route Treatment days day MIA 0.3 mg (6) PG-007 3 mg/kg 30, 35, 39, 45and 58 50 MIA 0.3 mg (6) P75NTR-Fc 0.3 mg/kg 30, 35, 39, 45 and 58 50MIA 0.3 mg (6) P75NTR-Fc 1 mg/kg 30, 35, 39, 45 and 58 50 MIA 0.3 mg (6)P75NTR-Fc 3 mg/kg 30, 35, 39, 45 and 58 50 MIA 0.3 mg (6) Human IgG FC30, 35, 39, 45 and 58 fragment full length 50 protein MIA 0.3 mg (3) PBS30, 35, 39, 45 and 58 50 Naïve (6) None None 58

Body weight was regularly recorded through the study and assessment ofspontaneous pain was measured at weekly intervals.

As anti NGF anti-neurotrophins have been shown to cause rats to scratcharound the face and neck, animals were regularly observed for signs ofskin lesions and any lesion scores recorded, particularly after day six,to ensure that we were not reaching the humane clinical end-point asdetermined in previous studies (animals will be killed if the maximumarea of total skin lesions exceeds 10 cm² or if any one lesion wasgreater than 2 cm×3 cm in size and became deeper and wet with no signsof healing within a 24 hour period).

On the terminal day a blood sample was taken by cardiac puncture andplasma prepared. The knees were removed and placed in 10% neutralbuffered formal saline and processed for histology.

Randomisation of Treatment(s)

Injection of MIA

Randomisation was carried out so that either the left or right knee ofeach rat was injected with MIA (with the contralateral knee from eachrat injected with ETF-PBS). The allocation of which knee received MIA orsaline for each rat was produced using a random number generator inMicrosoft Excel for the Mac (Version 14.1.1). Personnel who had nocontact with the animals carried out the randomisation procedure andallocation (see Appendix 1 for the schedule). Two 7 ml polypropylenevials were labelled for each animal to denote the left or right knee(total of 66 vials). Two people (one scoring and checking to the masterrandomisation sheet and one aliquoting the solution for theintra-articular injection) prepared the 66 vials. The aliquoting wascarried out in sequence so that the MIA vials were filled first followedwith the remaining vials being filled with ETF-PBS (this was thecontralateral knee vial for each animal). The vials for the naïveanimals were left empty.

Dosing of Test Agents

Animals were randomised into treatment groups of equal differences inthe nociceptive behaviour of the knee that had been treated with MIAcompared to the knee treated with vehicle control. This was performed onday 28 following the induction of OA.

Animal Procedures

Intra-Articular Injection of the Knee

Rats were anaesthetised by inhalation of Isoflurane using a BoylesApparatus. The hairs on both knees of each animal were clipped and theknees swabbed with ethanol. Each knee was injected through theinfra-patellar ligament with 50 μl of either 0.3 mg MIA in ETF-PBS orETF-PBS alone using a 0.5 ml sterile Becton Dickinson Micro-Fine insulinsyringe with an attached 27 G needle. The six naïve animals wereanaesthetised and the hairs on both knees were clipped only. Throughoutthe study in vivo scientists were blind to the treatment status of allanimals.

Dosing of Test Agents

Test agents were dosed by the subcutaneous route in the scruff of theneck or flank using a dose volume of 5 ml/kg.

Assessment of Spontaneous Pain

Spontaneous pain was determined for each animal at weekly intervals bymeasuring the weight bearing of the left and right hind limbs using anIncapacitance tester (Linton Instruments, U.K.). Rats were placed in anappropriately sized perspex animal box on the incapacitance tester sothat their hind feet sat on separate sensors. The size of the box chosenwas based on the body weight of the rat (small rat holder for rats up to450 g and large rat/guinea pig holder for rats over 450 g), whichallowed the rat to sit comfortably without being squashed, but similarlydid not offer too much space so that the rat could turn around. Once therat was steady and calm, the weight bearing of each limb was recordedover 5 seconds and the average force in grams exerted by both hind limbswas recorded. The weight distribution of the hind paws was determinedfive times for each rat at each time point, and the mean of the fivereadings calculated. The individual weight bearing data was convertedinto a weight distribution by dividing the weight of the right limb bythe total weight for both hind limbs.

Terminal Blood Sample

Terminal blood samples were taken by cardiac puncture under Isofluraneanaesthetic with a Terumo 2 ml syringe and 21 G needle that had beenflushed with 1% potassium EDTA in MilliQ water. The blood collected wasput into 2 ml polypropylene tubes. Animals were then killed by cervicaldislocation.

Plasma Preparation

Terminal blood samples were centrifuged at 2700×g for 10 minutes and theplasma aliquoted into polypropylene tubes (four aliquots per animal) andfrozen at −80° C.

Removal of the Rat Knees

The skin on the lower leg was removed and the muscle bundles separatedfrom the bones but left intact with the knee. The femur, fibula andtibia were severed and the knee with attached muscle was removed andplaced in approximately 80 ml of 10% neutral buffered formalin in a 125ml screw cap container. The knees were kept in buffered formalin from 48to 72 hours before being processed for histological analysis.

Processing for Histology

Tissue samples were prepared for light microscopy using standardprocedures and carried out externally at the University of Cambridgeveterinary school. Briefly, after the knee joints were fixed, thetissues were rinsed with PBS and subsequently decalcified in 8% formicacid for 10 days. Following decalcification the tissue was processedusing a Shandon Citadel tissue processer which dehydrated the tissuethrough a series of graded ethanol concentrations (six changes of 4 hourranging from 75% to 100% ethanol), rinsed with 100% chloroform (threechanges of 4 hour) and finally embedded into molten paraffin wax to forma tissue block (two changes of 4 hour). The tissues were embedded intocassettes using a Surgipath PEC 3001 machine with molten paraffin wax.The tissue was processed as soon as possible after collection tominimise damage caused from the formalin fixation so that tissuesections could be potentially used for immunohistochemistry.

At least four 10 μm sections were cut from each rat knee tissue blockusing a Leica RM2135 wax microtome. Two sections were placed on a glassslide and the slides were processed for standard Haematoxylin and Eosin(H&E) and Safranin O fast Green (Safranin O F/G) staining using anautomated linear staining machine (Leica ST4040). Briefly, the linearstaining machine has 23 reagent stations and four water stationsarranged in a configuration for staining slides, which follow a standardH&E or Safranin O F/G protocol. For H&E slides were exchanged betweenthe 27 stations after one minute in each and follow a xylene, ethanol,water, haematoxylin, water, acid alcohol, water, eosin, water, ethanoland xylene series. (all solvents from Fisher Scientific and H&E fromLeica). The sections were allowed to air dry, were mounted and coveredwith a coverslip ready to be viewed under a microscope.

Image Analysis

Slides were viewed at either times 2, 4 or 10× magnification using anOlympus AX70 microscope using Image-Pro Plus image analysis software(suite v7.0, Media Cybernetics, U.K.). An initial informal analysis ofthe stained tissue sections for OA-like features was performed to showthe overall gross changes of the medial knee joint of each rat.

Image analysis was conducted on the SO/FG slides using the ×2 objectivewhere the total area of the cartilage (in mm²) was determined for allthe rat knees (MIA and ETF-PBS treated knees). The depth of thecartilage layer (a minimum of 6 measurements was made per medial kneejoint) and this was compared to the depth of the sub-chondral bone fromthe same knee joint.

Data Analysis

Image analysis (such as cartilage area) and pain assessment data (forexample, weight distribution imbalance) was analysed for each animalusing classical statistics. Multiple measurements were collected andaveraged from animals treated with either p75-NTR-Fc (at differentdoses) or PG-007 and values were compared to control animals using theappropriate classical statistical tests. For all analyses, p<0.05 wastaken to indicate statistical significance.

Results

Body Weight

The body weights of the rats in the different groups were compared totest for differences in the sizes of the animals. At day 0, there was nostatistically significant difference between the body weights of theanimals in the different treatment groups (p=0.76 n.s., one-way ANOVA,see FIG. 30).

The mean body weight between the six treatment groups (rats treated withp75NTR-Fc or PG-007 compared to rats treated with control antibody) wasnot statistically significant from each other during the course of thestudy (p=n.s., two way ANOVA, see FIG. 31).

Spontaneous Pain Measurements

Effects of p75NTR-Fc and PG-007 on the Spontaneous Pain Provoked by OA

Spontaneous pain was assessed using an incapacitance tester to measurethe distribution of weight through the rear limbs. Assessments were madeat baseline (except for group 7, naïve animals) and again on days 15, 21and 28 following the injection of MIA into one knee (contralateral kneeswere injected with ETF-PBS). The data is shown in FIG. 32 and isillustrated as the proportion of the total weight over the rear limbsthat are being supported by the MIA-treated rear limb. For naïve animalsthe proportion of weight that is passing through the left rear limb isshown.

For all the animals there was no statistically significant differencebetween the proportion of weight on the treated rear limb and thetheoretical expectation of 0.5 (even distribution across both rear limbsat baseline on day 0 (p=0.379, Kruskal Wallis test; see FIG. 32). By day28, all five treatment groups were significantly different to thetheoretical mean of 0.5 and were essentially taking the weight of theirMIA injected knee (see FIG. 32). Based on previous studies thisindicated that the extent of OA pathology was significant enough tostart dosing the rats.

Dosing was started in rats with the respective treatment regimen fromday 30 onwards until the end of the study on day 56 (treatment every 5days via the subcutaneous route). During the course of the study furtherassessment of spontaneous pain were measured (made on days 35, 42, 49and 56, see FIG. 4). Throughout the study up until day 49, animalstreated with control IgG Fc remained significantly different between theproportion of weight on the treated rear limb and the theoreticalexpectation of 0.5, indicating that the MIA treated knee was causingpain to the animal (see FIG. 33). In contrast, in animals treated withanti-NGF PG-007 and those treated with p75NTR-Fc (from 0.3 to 3 mg/kg)there was no significant between the proportion of weight on the MIAtreated rear limb and the theoretical expectation of 0.5 (ie evendistribution of weight on the rear limbs) as time progressed.

Histological Changes in the Knee Following Mia-Induced Oa

Effects of p75NTR-Fc and PG007 on the Regression of OA

Knees injected with MIA from animals treated with control antibodiesshowed areas of chondrocyte degeneration, which in some areas has led tofull cartilage loss (FIG. 34). This is highlighted by the associatedloss of Safranin O fast green staining in the cartilage. In addition,there were significant pathological changes in the sub chondral boneunderlying the areas of the greatest cartilage damage (FIG. 34). Thereis a spectrum of changes in the subchondral bone, which reflect bothreactive changes and progression driven from remodelling through tosclerotic changes (FIG. 34).

Animals treated with 3 mg/kg PG-007 for 28 days after significantpathology has been established (identified by a pain assessment wherethe proportion of weight on the treated rear limb and the theoreticalexpectation of 0.5 were significantly different) showed significanthistological changes compared to animals treated with control antibodywith loss of the overall integrity of the rat knee and no histologicalevidence of efficacy (FIG. 35). This pathology is consistent withassociated loss of cartilage and bone marrow and evidence of bonenecrosis, which overall leads to an overall rapid progression and insome animals a worsening of the OA compared to animals treated withcontrol antibodies (FIG. 35). In contrast, animals treated withp75NTR-Fc (0.3 to 3 mg/kg) exhibit a very different histopathology andthere is evidence of significant chondroprotection and an associatedsignificant increase in subchondral bone pathology compared to animalstreated with control antibodies, which leads to an overall reducedseverity of the bone pathology and OA (FIGS. 36-38). This isphenotypically represented by a reduced myeloid expansion in the bonemarrow, which diminishes the inflammatory mediated osteolytic drive.

Animals treated with the lower doses of p75NTR-Fc (0.3 and 1 mg/kg) showthe most marked changes in the histopathology (see FIGS. 37 and 38), andin both treatment groups the pathology was almost at normal levels.Taking into consideration the histological results at 3 mg/kg p75NTR-Fc(see FIG. 36) the overall profile suggests that p75NTR-Fc exhibits a“bell-shaped” dose response curve.

Histopathology Assessment

Histopathology was performed on Safranin O Fast Green stained sectionsof decalcified knees. In general, the quality of both tissue processingand staining was excellent, with only three slides rejected on qualitygrounds. The slides were organised into treatment groups and so theinitial pathology assessment was not blinded. However, for the purposesof grading, all groups (with the exception of vehicle) wererandomised—using a simple random number sequence—in order to reduceobserver bias and diagnostic drift.

Descriptive Cartilage Histopathology Phenotypes

Control Group

There was a clear distinction between paired slides. Slides with mostmarked cartilage pathology are: 4RA, SLA, 6LB, 10RA, 11LB (insufficientcartilage to grade on slide 11RA) and 12RB. The majority of cartilagechanges were full thickness erosions with obvious peripheral chondrocytenecrosis.

Group 4—PG007, 3 mg/kg

There was a clear distinction between paired slides. Slides with mostmarked cartilage pathology are: 1RA, 2RB, 3LB, 34LA, 35LA, 36RB.Overall, the cartilage pathology was histologically similar to thecontrol group.

Group 3—P75NTR-Fc, 0.3 mg/kg

The distinction between paired slides was not so marked compared to thecontrol group. Administration of P75NTR-Fc was associated with markedinhibition of MIA-induced cartilage pathology—indeed near normal in twocases. Three slides were rejected from this group on quality grounds.

Group 1—P75NTR-Fc, 1.0 mg/kg

The distinction between paired slides was not so marked compared to thecontrol group. Slides with most marked cartilage pathology (althoughmodest compared to control lesions) are: 7LA, 8LA, 13RB, 15RA, 31RB,33LB. Administration of P75NTR-Fc was associated with marked inhibitionof MIA-induced cartilage pathology.

Group 2—P75NTR-Fc, 3.0 mg/kg

This group presented with marked variability in terms of efficacyeffects on cartilage pathology—with a spectrum of effects fromcontrol-type lesions to those resembling Group 1 (50%). Slides with themost marked cartilage pathology are: 16LB, 17RA, 18RB, 22RB, 23LA and24LA. Administration of P75NTR-Fc at the top dose did not show theunequivocal effects associated with 0.3 and 1.0 mg/kg.

Histopathology Grade Criteria

Allocation of grade is based upon the most frequent lesion observedwithin each anatomical zone. Based upon the initial assessment, keyhistological features were identified in the control group and whichshowed evident changes following 1.0 mg/kg P75NTR-Fc administration.Grade criteria were those which have been employed in previous MIA studypathology assessment (MLF).

Cartilage Pathology

Many publications detail the use of the Mankin Score for grading humancartilage pathology. The original Mankin Score (or HHGS score) rangesfrom 0 (normal) to 14 (severe pathology) and suffers from markedinter-observer bias. In addition, this scoring system underestimates theinvolvement of pannus pathology and often conflates early changes withnormal variation. Importantly, the Mankin Score expands the grade rangefor moderate to severe pathology, and markedly conflates mild tomoderate changes. Thus, the Mankin Score, applied directly, hasquestionable utility in pre-clinical studies as it rarely describes apharmacodynamic range and is insensitive to the differences in cellturnover between rodents and man. The grading system used in this studyis a modification of the OARSI system using the descriptive scheme ofPelletier:

0—no significant abnormalities; 1—minimal superficial fibrillation;2—superficial erosion; loss of superficial zone chondrocytes; 3—deepzone erosion; 4—full thickness erosion; multi-focal exposure ofsub-chondral bone, <50% area; 5—loss of cartilage plate; detritic formsin articular space, >50% area

Sub-Chondral Bone

The sub-chondral bone plate reacts to loss of cartilage integrity acrossthe spectrum of cartilage grades before undergoing degenerativechanges—essentially osteolysis mediated. 0—no significant abnormalities;1—superficial zone disorganisation (erratic osteoid zones); 2—fullthickness disorganisation; 3—multi-focal osteolysis; 4—multi-focalosteopenia; 5—confluent osteopaenic zones

Stromal Cavities

The stromal cavities are the marrow cavities within and immediatelydistal to the subchondral plate. 0—no significant abnormalities;1—minimal osteolysis; 2—moderate osteolysis; 3—marked osteolysis withevidence of multiple lytic pits in majority of cavities;4—post-osteolytic fusion of cavities; 5—fused cavities breachsub-chondral bone and/or periosteum

Cancellous Bone

The cancellous bone system undergoes reactive changes in minimalcartilage pathology due to changes in joint biomechanics—progressing toa more degenerative phenotype secondary to inflammation

0—no significant abnormalities; 1—multi-focal areas of superficial boneresorption/osteolysis; 2—multiple areas of marked osteolysis; 3—multipleareas of osteolysis with loss of normal tide-mark zonation;4—multi-focal zones of osteolysis with evident osteocyte loss;5—cancellous bone breach with/without bone fusion

Bone Marrow Hyper-Cellularity

0—no significant abnormalities; 1—multiple focal condensed foci of cellswithin marrow stroma; 2—multiple focal condensed foci of cells withinmarrow stroma and periosteal pits; 3—diffuse marrowhyper-cellularity—zonal; 4—expansion of bone marrow into stromalcavities; 5—breach of marrow compartment into sub-chondral plate orperiosteal with/without pannus mixing

Bone Sclerosis

Bone sclerosis is usually seen as areas of fibroplastic expansion fromthe stromal cavities. 0—no significant abnormalities; 1—occasional,small, foci; 2—multiple foci; 3—multiple confluent foci; 4—multipleconfluent foci with associated bone degeneration; 5—multiple confluentfoci with bone cysts

Osteoblast Hyperplasia

0—no significant abnormalities; 1—multiple osteoblast plates; 2-multiplehypertrophic osteoblast plates; 3—obvious osteoblast palisading;4—hypertrophic osteoblast plates-zonal; 5—hypertrophic osteoblastplates—multi-zonal.

Histology Grade Results (Most Severe Cartilage Pathology Sample fromPaired Sets Plotted)

Cartilage Pathology

Administration of PG007 showed no histologically significant efficacyeffects. P75NTR-Fc showed marked efficacy on chondroprotection at 0.3and 1.0 mg/kg. The efficacy profile at 3.0 mg/kg was more variable—with50% of the group showing overlay with the control group (FIG. 39).

Sub-Chondral Bone

Administration of PG007 was associated with histologically significantincrease in sub-chondral bone pathology compared to the controlgroup—similar to the profile observed with 3.0 mg/kg P75NTRFc. Bycontrast, administration of P75NTR-Fc at 0.3 and 1.0 mg/kg wasassociated with marked improvement in sub-chondral bone histology (FIG.40)

Stromal Cavities

Administration of PG007 was not associated with any histologicallysignificant efficacy effects. By contrast, administration of P75NTR-Fcat all does was associated with reduction in osteolytic pathology andexpansion of stromal cavities, although the effects at 0.3 and 1.0 mg/kgwere most marked—both groups reducing pathology to almost normal levels(FIG. 41).

Cancellous Bone

Administration of PG007 was associated with cancellous bone pathologysimilar to controls—with two samples exceeding control ranges. Bycontrast, P75NTR-Fc markedly reduced cancellous bone pathology at the0.3 and 1.0 mg/kg dose levels. P75NTF-Fc at 3.0 mg/kg presented a morevariable profile, with the majority of samples showing overlay with thecontrol group (FIG. 42).

Bone Marrow Hyper-Cellularity

Administration of PG007 was associated with expansion of myeloid cellsin the bone marrow, with four samples either at the top or exceeding thecontrol range. P75NTR-Fc reduced marrow cellularity—with reduced myeloidexpansion being a prominent feature at the 0.3 and 1.0 mg/kg doses. The3.0 mg/kg dose, although showing a trend towards inhibition, showedoverlay with low level responders in the control group (FIG. 43)

Bone Sclerosis

There were no histologically significant differences between the studygroups—although the P75NTR-Fc samples did show a clustering towards thetop end of the control range (FIG. 44).

Osteoblast Hyperplasia

Administration of P75NTR-Fc at 0.3 and 1.0 mg/kg was associated withhistologically significant osteoblast proliferation—with palisading andosteoblastic plate formation being especially prominent in theepiphyseal zone, at the sub-chondral bone/cartilage zone. In addition,although not graded, there were prominent ‘fibroblast-like’ cells inthis zone. PG007 and P75NTR-Fc (3.0 mg/kg) were histologically similarto controls (FIG. 45).

Adverse Effects

Two rats treated with 3 mg/kg PG-007 developed skin lesions. Very minorskin lesions began to appear around the face, neck and shoulder regionin some rats by day 45 (15 days following treatment) but became moreapparent from day 49 (19 days following treatment) onwards in one rat.These lesions were not associated with the injection site. Rats werechecked regularly and the number, size and extent of each lesion wasquantified to ensure we remained within the humane clinical end-point ofthe study. No rats treated with p75NTR-Fc at any concentration developedskin lesions.

Discussion

Histopathology

PG007 was not associated with histological evidence of efficacy. Incontrast, P75NTR-Fc at 0.3 and 1.0 mg/kg was associated withhistologically significant chondroprotection, with reduced severity ofbone pathology and osteolysis-mediated bone erosion. The finding ofreduced bone marrow hyper-cellularity (notably myeloid expansion)supports this phenotype, and suggests that limitation of aninflammation-mediated osteolytic drive is involved in the efficacyresponse. Importantly, these doses were also associated with anexpansion of the osteoblast pool—suggestive that the mechanism ofefficacy of P75NTR-Fc is a dual mechanism, limiting osteolysis (thuslimitation of myeloid cell activation) whilst expanding the osteoblastpool (thus augmenting the mesenchyme cell pool). The histologicalprofile of the 3.0 mg/kg P75NTR-Fc group suggests that P75NTR-Fcexhibits a ‘bellshaped’ dose response curve. Although in many respectsthe histological profile of 3.0 mg/kg P75NTR-Fc and PG007 are similar,the exact phenotype suggests that the osteolytic drive of P75NTRFc isless than that of PG007 on a dose equivalency basis. Classically,histopathology assessment of MIA models is based upon a linearrelationship from primary cartilage pathology resulting in reactive bonechanges. The bone marrow changes are often deemed consequential,secondary to a reactive synovitis arising from biomechanical changes dueto cartilage loss. There is accumulating data from osteoimmunologysuggesting that this linear relationship is naïve, and that the bonecompartment can exert an important role in conditioning both thetemporal development and phenotype of the cartilage response. Indeed,this research supports imaging data from human studies which show boneremodelling even in minimal grade cartilage lesions. Thus it is likelythat the cartilage and sub-chondral bone should be viewed as anintegrated functional unit—with the latter providing conditioning tosupport chondrocyte proliferation and survival and so influencingstructural outcomes in osteoarthritis. The data from the present studyoffers support to the hypothesis that manipulation of the NGF pathway,whilst offering possibilities for reducing osteolysis, also may augmentmesenchyme cell mediated osteoprotection—and thus suppressing the drivefrom superficial chondronecrosis to deep zone lesions and hence fullthickness cartilage erosions and cartilage loss.

CONCLUSIONS

In this study the effect of a therapeutic dosing regimen using p75NTR-Fc(from 0.3 to 3 mg/kg) was investigated to determine whether regressionof the OA pathology following MIA injection could be observed. OA wasallowed to develop for 30 days before dosing commenced, which wasidentified by pain measurements being significantly different from thetheoretical mean of 0.5.

Animals treated with lower doses of p75NTR-Fc (0.3 and 1 mg/kg) wereboth analgesic and efficacious and showed significantly less OApathology compared to animals treated with control antibodies. Incontrast, despite rats treated with 3 mg/kg PG-007 showing analgesiathis was not associated with histological evidence of efficacy and anyimprovement in the OA pathology. Animals treated with the higher dose ofp75NTR-Fc (3 mg/kg) were analgesic however; there were somehistopathological similarities to animals treated with 3 mg/kg PG-007.

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description and accompanyingfigures. Such modifications are intended to fall within the scope of theappended claims. Moreover, all embodiments described herein areconsidered to be broadly applicable and combinable with any and allother consistent embodiments, as appropriate.

Various publications are cited herein, the disclosures of which areincorporated by reference in their entireties.

1-23. (canceled)
 24. A method of treating osteoarthritis in a subjectsuffering therefrom, the method comprising administering to the subjecta therapeutically effective amount of a p75NTR neurotrophin bindingprotein (p75NTR(NBP)).
 25. The method according to claim 24, whereintreatment of osteoarthritis includes relief from the symptoms ofosteoarthritis.
 26. The method according to claim 25, wherein relieffrom the symptoms of osteoarthritis includes reduction in pain,inflammation, swelling, tenderness, joint stiffness or increase in jointmobility, or any combination thereof.
 27. The method according to claim24, wherein treatment of osteoarthritis includes slowing or arrestingdisease progression.
 28. The method according to claim 24, whereintreatment of osteoarthritis includes reversal of disease progression,regrowth of cartilage, curative treatment, or any combination thereof.29. The method according to claim 28, wherein disease progression isdetermined by rate of cartilage loss or regrowth.
 30. The methodaccording to claim 24, wherein treatment of osteoarthritis includesprophylactic treatment.
 31. The method according to claim 24, whereinthe p75NTR(NBP) is connected to one or more auxiliary molecules.
 32. Themethod according to claim 31, wherein the one or more auxiliarymolecules are selected from the group consisting of transferrin or aportion thereof, albumin or a portion thereof, an immunoglobulin Fc or aportion thereof, and a polyethylene glycol polymer chain.
 33. The methodaccording to claim 31, wherein the p75NTR(NBP) is connected to the oneor more auxiliary molecules via one or more linkers.
 34. The methodaccording to claim 32, wherein the p75NTR(NBP) is connected to animmunoglobulin Fc or a portion thereof.
 35. The method according toclaim 24, wherein the p75NTR(NBP) comprises an amino acid sequenceaccording to SEQ ID NO:3.
 36. The method according to claim 24, whereinthe p75NTR(NBP) binds to any of NGF, BDNF, NT3 or NT4/5 with a bindingaffinity (Kd) of about 5 pM to about 5 nM as measured by surface plasmonresonance at 20° C.
 37. The method according to claim 24, wherein thep75NTR(NBP) is administered to the subject separately, sequentially, orsimultaneously in combination with a second pharmacologically activecompound.
 38. The method according to claim 37, wherein the secondpharmacologically active compound is one of an opioid analgesic, anonsteroidal anti-inflammatory drug (NSAID), a barbiturate sedative, abenzodiazepine having a sedative action, an H1 antagonist having asedative action, a sedative such as glutethimide, meprobamate,methaqualone or dichloralphenazone; a skeletal muscle relaxant, an NMDAreceptor antagonist, an alpha-adrenergic, a tricyclic antidepressant, ananticonvulsant, a tachykinin (NK) antagonist, particularly an NK-3, NK-2or NK-1 antagonist; a muscarinic antagonist, a COX-2 selectiveinhibitor, a coal-tar analgesic, in particular paracetamol; aneuroleptic a vanilloid receptor agonist or antagonist, abeta-adrenergic, a local anaesthetic, a corticosteroid, a 5-HT receptoragonist or antagonist, a 5-HT2A receptor antagonist, a cholinergic(nicotinic) analgesic, Tramadol®, a PDEV inhibitor, a cannabinoid,metabotropic glutamate subtype 1 receptor (mGluR1) antagonist, aserotonin reuptake inhibitor, a noradrenaline (norepinephrine) reuptakeinhibitor, a dual serotonin-noradrenaline reuptake inhibitor, aninducible nitric oxide synthase (iNOS) inhibitor, anacetylcholinesterase inhibitor, a prostaglandin E2 subtype 4 (EP4)antagonist, a leukotriene B4 antagonist, a 5-lipoxygenase inhibitor, asodium channel blocker, or a 5-HT3 antagonist, and pharmaceuticallyacceptable salts and solvates thereof.
 39. The method according to claim24, wherein the p75NTR(NBP) is formulated for oral, sublingual, buccal,topical, rectal, inhalation, transdermal, subcutaneous, intravenous,intra-arterial, intramuscular, intracardiac, intraosseous,intrasynovial, intradermal, intraperitoneal, transmucosal, vaginal,intravitreal, intra-articular, peri-articular, local or epicutaneousadministration.
 40. A nucleic acid encoding the p75NTR(NBP) according toclaim
 24. 41. A replicable expression vector comprising the nucleic acidaccording to claim
 40. 42. A host cell expressing the p75NTR(NBP)according to claim
 24. 43. A pharmaceutical composition, comprising thep75NTR(NBP) according to claim 24 and a pharmaceutically acceptablecarrier, an excipient, or a combination thereof.
 44. A kit comprising:a) the p75NTR(NBP) according to claim 24; and b) instructions for theadministration of the therapeutically effective amount according toclaim 24 to a subject suffering from osteoarthritis.